Blood-Brain Barrier Model

ABSTRACT

A method of creating a multicellular blood-brain barrier model is disclosed. In one embodiment, the method comprises culturing primary brain microvascular endothelial cells or embryonic stem cell-derived endothelial cells upon a permeable support in the presence of neural progenitor cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/218,123, filed on Aug. 25, 2011, which is a divisional application ofU.S. application Ser. No. 11/766,633 filed on Jun. 21, 2007, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.60/816,033 filed on Jun. 23, 2006. Each of these applications isincorporated by reference herein.

STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AA013834 andNS052649 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

The blood-brain barrier (BBB) is composed of a specialized class ofendothelium that forms a cellular barrier between the bloodstream andthe interstices of the adult brain. By restricting non-specific flux ofblood-borne constituents, the BBB plays an important role in maintainingparenchymal homeostasis, and strictly regulates transport of ions, smallmolecules, proteins, and cells into and out of the brain. The BBBaccomplishes these tasks because its unique endothelium is endowed byepithelial-like tight junctions joining adjacent endothelial cells,lacks fenestrae, and possesses a rich array of molecular transportsystems. Although the endothelium is the principle determinant ofbarrier function, perivascular non-endothelial cells in the localmicroenvironment have been shown to make significant contributions.Astrocytes (Stewart and Wiley 1981; Risau et al. 1986b; Janzer and Raff1987), neurons (Tontsch and Bauer 1991) and pericytes (Balabanov andDore-Duffy 1998; Ramsauer et al. 2002) have all been demonstrated toprovide cues that result in the unique BBB endothelial phenotype.

Although the inductive properties of the aforementioned brain cell typeshave been confirmed through a multitude of in vivo and in vitro studies,the cell type(s) responsible for early embryonic BBB induction have notbeen distinguished. The developmental timecourse of embryonic BBBformation differs between species, but it is generally well acceptedthat the onset of BBB development begins prenatally and is followed by agradual maturation to full BBB function (Bauer and Bauer 2000;Engelhardt 2003). For example, in rodents, vascular fenestrae disappear,pinocytosis decreases, and vessels decrease in diameter betweenembryonic days E11 and E17 (Bauer et al. 1993; Stewart and Hayakawa1994; Bolz et al. 1996). The onset of tight junction formation isdetectable from day E15, and tight junctions continue to increase incomplexity through postnatal day P1 (Butt et al. 1990; Schulze and Firth1992; Bauer et al. 1995; Kniesel et al. 1996; Nico et al. 1999). Duringthis time, the transendothelial electrical resistance (TEER) of pialvessels is intermediate between peripheral vessels and the adult BBB(Butt et al. 1990; Schulze and Firth 1992; Bauer et al. 1995; Kniesel etal. 1996; Nico et al. 1999). A combination of the aforementionedattributes serves to restrict passage of protein into the embryonicbrain (Risau et al. 1986a; Bauer et al. 1995; Dziegielewska et al.2000), while a gradual decrease in BBB permeability to small tracerssuch as inulin and sucrose begins during embryonic development andcontinues postnatally (Ferguson and Woodbury 1969). Finally, transporterexpression at the BBB also evolves from embryonic to postnatal stages asa result of changing nutritional needs (Johanson 1989; Gerhart et al.1997).

The early embryonic developmental timecourse for the BBB raises thequestion as to what inductive factors or cell types drive theendothelial differentiation process. As mentioned above, astrocytes havelong been linked with induction of BBB properties by in vitro and invivo experiments (Stewart and Wiley 1981; Risau et al. 1986b; Janzer andRaff 1987). However, angiogenic vessels invade the immature embryonicneural environment and begin establishing BBB characteristics well inadvance of the onset of gliogenesis as defined by the presence ofGFAP-positive astrocytes in rodent brain (E18, (LeVine and Goldman1988)). In addition, the developing BBB vessels have littleextracellular matrix with few astrocyte contacts even just days prior tobirth (Caley and Maxwell 1970). In fact, for rodents, much of theastrocyte generation takes place postnatally during which time theastrocyte sheath that surrounds mature brain capillaries is developed(Johanson 1989). Therefore, it is unlikely that astrocytes function inthe early BBB induction process, but instead other cell types may beresponsible for the early onset of BBB properties.

NPC are a major cell type in the developing embryonic brain, and it wasrecently reported that the differentiation and morphology of NPC areinfluenced by endothelial cells (Shen et al. 2004). In co-culture withendothelial cells, NPC show reduced neurogenesis and elevatedself-renewal (Shen et al. 2004). Neural progenitors have also beenobserved in contact with early postnatal blood vessels, and this wasimplicated as an early stage in astrocyte differentiation (Zerlin andGoldman 1997). In addition, when endothelial cells and neural stem cellsare grown in direct contact, it was shown that the adult neural stemcells could even produce progeny that exhibited an endothelial phenotype(Wurmser et al. 2004). Finally, adult neural stem cells are often foundlocalized in perivascular spaces of the brain such as the subventricularzone and hippocampus, and it is thought that the vasculature is animportant part of the stem cell niche (Doetsch 2003b).

Given the cellular interplay in the endothelial cell to NPC direction,we examined herein whether NPC could also influence brain endothelialcell phenotype. In this specification, we demonstrate that NPC isolatedfrom the E14 embryonic brain induced BBB properties in an in vitro modelconsisting of primary rat brain microvascular endothelial cells inco-culture with NPC. We disclose an improved BBB model and method forexamining the permeability of the BBB to test compounds.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention is a method of creating amulticellular blood-brain barrier model, comprising the step ofculturing brain microvascular endothelial cells upon a permeable supportin the presence of neural progenitor cells, wherein the cultured neuralprogenitor cells differentiate into mixtures of astrocytes, neurons, andoligodendrocytes such that a multicellular blood-brain barrier model iscreated.

In one embodiment the endothelial cells are isolated from mammalianbrain capillaries. In another embodiment, the endothelial cells arederived from isolated embryonic stem cells.

In a preferred embodiment, the endothelial cells form a monolayerwherein the cells are confluent and express a preferable TEER of 20-50,most preferably 30-40 Ohm×cm², before exposure to the neuroprogenitorcells (NPCs) and greater than 100 Ohm×cm² after exposure to theneuroprogenitor cells (NPCs). In a most preferred embodiment, the TEERof the BBB is 100-250 Ohm×cm².

In one embodiment, the neural progenitor cells are isolated frommammalian cortices and are digested with ACCUTASE™ enzyme mixture. Inone embodiment the neural progenitor cells are grown as free-floatingneurospheres before exposure to the endothelial cells. In oneembodiment, the neural progenitor cells are pre-differentiated beforeexposure to endothelial cells. In one embodiment the neural cells areremoved after the endothelial cells are confluent and express a TEERgreater than 100 Ohm×cm².

The invention is also a blood-brain barrier model comprising at leastthree components within a liquid-containing vessel. The first componentcomprises a confluent layer of brain microvascular endothelial cells orembryonic stem cell-derived endothelial cells, the second componentcomprises a permeable membrane support, wherein the first componentforms a layer on the second component, and the third component compriseseither (a) undifferentiated neural progenitor cells that aredifferentiated after contact with the first component to be a mixture ofastrocytes, neurons and oligodendrocytes or (b) neural progenitor cellsthat have been pre-differentiated before contact with the firstcomponent to be a mixture of astrocytes, neurons and oligodendrocytes.The first and second components form a barrier between a top and abottom chamber of the vessel and the third component is placed in thebottom chamber of the vessel. The third component may be in directcontact with the first and second component or may be separated byfluid.

Preferably the endothelial cells form a monolayer wherein the cells areconfluent and express an initial TEER of 20-50, most preferably 35Ohm×cm², before exposure to neuroprogenitor cells (NPCs). After exposureto neuroprogenitor cells (NPCs), most preferably the TEER is 100-250Ohm×cm².

In one embodiment, one may construct the model described above andremove the neural cells after a TEER of greater than 100 Ohm×cm² hasbeen obtained.

In another embodiment, the invention is a method of analyzing theblood-brain barrier permeability characteristics of a model compound,comprising the steps of exposing a model compound to the blood-brainbarrier model and measuring the permeability of the barrier model to thecompound.

Other features of the present invention will become apparent afterreview of the specification, claims and drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: In vitro model of the BBB. Primary BMEC were cultured for 2 daysin the presence of puromycin to yield a nearly 100% pure BMEC monolayeron the collagen IV/fibronectin-coated filter support. On DIV 2 BMEC wereswitched to puromycin-free medium. After reaching confluence on DIV 3,NPC were added to the lower compartment in the presence or absence ofmitogens (FGF-2, EGF). The resulting co-cultures were used to assess NPCeffects on the BBB properties of the BMEC monolayer.

FIG. 2: Influence of NPC co-culture and NPC-conditioned media on BMECTEER. BMEC were cultured as follows: 1. in the absence of NPC (control),2. in the presence of differentiating NPC (+NPC −mitogens), 3. in thepresence of 24 h pre-differentiated NPC (+NPC 24 h prediff), 4. inNPC-conditioned medium generated by 24 hour of NPC culture inmitogen-free differentiation medium (NPC conditioned −mitogens), 5. inthe presence of NPC in mitogen-containing medium (NPC +mitogens), and 6.in 24 hour conditioned mitogen-containing medium from dividing NPC(NPCconditioned +mitogens). The TEER was monitored after 24 hours for eachculture condition and expressed as normalized % of control TEER values.The mitogen-containing conditions (5 and 6) were independentlynormalized to a mitogen-containing BMEC monoculture control. Thestatistical significance is given with p<0.001 (**) and p<0.03 (*) asdetermined by the unpaired Student's t-test. Triplicate cultures wereanalyzed for each condition and results display mean±SD. The results arerepresentative of 8 independent experiments where the increase in TEERin the presence of differentiating NPC (2) ranged from 17-53%.

FIG. 3: Tight junction organization and cell morphology of BMEC in thepresence (A, C, E) or absence (B, D, F) of differentiating NPC. After 24hours of co-culture in mitogen-free medium, the BMEC were probed forZO-1 (A, B), occludin (C, D) or claudin 5 (E, F). Note the tightjunction protrusions perpendicular to the cell-cell borders thatrepresent frayed junctions. An example denoted by the arrow is enlargedand shown in the inset of panel B. Representative fields are shown toillustrate the differences in junctional morphology. See Table 1 for thecorresponding quantitative data. Scale bar represents 50 μm.

FIG. 4: Influence of BMEC on NPC differentiation. NPC were fixed 24 hafter culture in presence or in absence of BMEC. Cell types weredetermined by indirect immunofluorescence using cell markers (βIIItubulin, GFAP, and nestin), and cells were counted. The statisticalsignificance is given with p<0.001 (***), p<0.01 (**), and p<0.03 (*) asdetermined by the unpaired Student's t-test. Results represent themeans±SD of 5 different microscope fields (˜1000 cells total).

FIG. 5: Influence of BMEC co-culture on NPC morphology. Merged images ofNPC that were probed for GFAP, nestin, and DAPI (panels A and B) or βIIItubulin, nestin, and DAPI (panels C and D). Panels A and C representdifferentiation in the absence of BMEC and Panels B and D in thepresence of BMEC. Arrows indicate small cell bodies and elongated thinprocesses (panels A and C) while arrowheads point to larger cell bodiesand short processes (panels B and D). Scale bar represents 20 μm.Figures are shown in grayscale. For color figures see Weidenfeller etal, 2007.

FIG. 6: Developing rat cortex at embryonic day E14. Panel A shows vonWillebrand factor-positive blood vessels in the developing cortex. ThevWF-positive vessels (several denoted by arrows) are localized inregions with large numbers of nestin-expressing cells and aredistributed from the pial surface to the ventricle. Panel B indicates ahighly βIII tubulin-positive region at the pial surface and homogenousdistribution of neurons throughout the NPC-positive ventricular zone.Panel C again indicates the distribution βIII tubulin-positive neuronsin addition to the absence of the tight junction protein occludin. PanelD illustrates that while the E14 rat cortex is highly nestin-positive,the astrocytic protein GFAP was not detected. P=pial surface;V=ventricle. Scale bar represents 400 μm. Figures are shown ingrayscale. For color figures see Weidenfeller et al, 2007.

FIG. 7: TEER measurements of BMEC in co-culture with NPC or astrocytes.NPC or postnatal astrocytes were co-cultured with confluent BMEC usingidentical mitogen-free culture medium, and the TEER timecourse wasfollowed for 5 days. Triplicate cultures were analyzed for eachcondition and the resistance measurements normalized to those of theuntreated control at time zero, and results display mean±SD. Statisticalsignificance between sample and control is indicated: p<0.006 (*),p<0.02 (**), and p<0.001 (***). The results are representative of 5independent co-culture experiments from multiple BMEC, NPC and astrocyteisolations. Percent increases denoted in the text refer to TEERincreases relative to the control at the time points of interest (24 or48 hours). The initial increase in TEER for the control BMEC culturesobserved at 24 hours was reproducible, and was a result of the reductionin serum content upon the switch to mitogen-free medium.

FIG. 8: TEER measurements of BMEC in co-culture with NPC-derivedastrocyte and neuron mixed cultures or postnatal primary astrocytes.Postnatal primary astrocytes, or predifferentiated NPC (differentiationprocess induced 6, 8, 10, 12, and 14 days prior starting the co-culture)were co-cultured with confluent BMEC using mitogen-free culture medium,and the TEER timecourse was followed for 7 days. Predifferentiated NPCwere prepared as follows: NPC were expanded after isolation for oneweek. Spheres were digested with ACCUTASE™ enzyme mixture, frozen down,and stored in liquid nitrogen until further use. 4 days prior inductionof differentiation, NPC were thawed and plated in NPC culture media toform spheres. The differentiation of spheres digested with ACCUTASE™enzyme mixture was induced by mitogen removal from the culture mediumand plating on poly L-lysine/laminin coated 12 well plates (2×10⁵cells/cm⁵). The medium was changed every three days. At 6, 8, 10, or 12days after initiation of differentiation in mitogen-free medium with 1%serum the filter with confluent BMEC was added. Triplicate cultures wereanalyzed for each condition and the resistance measurements normalizedto those of the untreated control at time zero, and results displaymean±SD.

FIG. 9: TEER measurements of BMEC in co-culture with co-differentiatinghuman and rat NPC. Co-differentiating rat and human NPC were co-culturedwith confluent BMEC using mitogen-free culture medium, and the TEERtimecourse was followed for 7 days. Human NPC were kept in culture for20 weeks. Spheres were chopped once a week and re-plated in 50% freshand 50% conditioned medium. Prior to plating the human NPC, spheres weredigested with ACCUTASE™ enzyme mixture and single cells were plated inaccordance with the rat NPC. Triplicate cultures were analyzed for eachcondition and the resistance measurements normalized to those of theuntreated control at time zero, and results display mean±SD.

DESCRIPTION OF INVENTION In General

Accurate reproduction of the in vivo blood-brain barrier (BBB) in an invitro setting has been a longstanding challenge in academia and theneuropharmaceutical industry. Issues of model quality includedifficulty, purity, functionality, reproducibility, high throughputcapacity, and accurate drug permeability prediction. To date, the mostsuccessful models usually include co-culture of primary brainmicrovascular endothelial cells (BMECs), which form the BBB in vivo,with primary brain astrocytes.

However, other brain cells such as neurons and neural progenitor cellshave been shown to help govern in vitro BBB properties. Thismulticellular composite is known as the neurovascular unit.Unfortunately, current methods require the independent isolation ofmultiple cell types (astrocytes and neurons in particular) to try andreproduce the neurovascular unit in vitro, and the quality of thesepreparations invariably differs from experiment to experiment and fromlaboratory to laboratory.

Our approach to improving both the in vivo character as well as thereliability of in vitro BBB models is using embryonic neural progenitorcells (NPCs) as source tissue. Embryonic neuroprogenitor cells (NPCs)(nestin-positive) are easily isolated and expand rapidly for up to sixweeks for rats and much longer for humans, leading to a largecomparatively homogenous cell stock (Ostenfeld et al., 2002; Ostenfeldand Svendsen, 2003.) neuroprogenitor cells (NPCs) can be stimulated todifferentiate into each of the major brain lineages, includingastrocytes, neurons, and oligodendrocytes (Ostenfeld and Svendsen, 2003)Thus, from just one cell type (and isolation), many of the important BBBeffector cells from the neurovascular unit can be simply generated as amixed culture.

It is an advantage of the present invention that the relativepercentages of the neurons and astrocytes can be controlled to createdesigner mixtures of brain cells that can be co-cultured with the brainmicrovascular endothelial cells. Several approaches can be used to tunethe “brain side” of the co-culture model. Essentially, the timing afterisolation from embryonic brains (time of expansion in culture withoutdifferentiation after isolation) influences the differentiationcapacities of neuroprogenitor cells (NPCs). Early on (for example, 1week [±1 day] for the rat cells) after isolation a higher ratio ofneuroprogenitor cells (NPCs) differentiate into neurons while laterinduction of differentiation (for example, 4 weeks [±2 days] for ratcells) results in higher numbers of astrocytes (Ostenfeld and Svendsen,2003).

In addition to this approach, chemical mediators can be used to directthe differentiation into the different NPC-derived cells. Asnonexhaustive examples, bone morphogenic protein 2 drives thedifferentiation process towards glial fate and cyclic AMP is suitablefor inducing the maturation (in vivo-like astrocyte) of astrocytes(Enkvist et al., 1996). In addition, to obtain higher neuron-containingcultures, retinoic acid can be used to trigger neuronal differentiation(Gallo et al., 2002). This is also possible by using mediators thattrigger the Wnt signaling pathway (Katoh, 2002). Taken together, thesemethods can be used to “tune” the brain side of the co-culture model,thereby tuning the response of the BMEC monolayer.

The BBB model of the present invention performs admirably in terms ofits permeability properties and outperforms astrocyte co-culture (seeExamples). Also, as described above, the model is likely morereproducible given that each experiment can rely on the same NPC stock.

From an important practical perspective, we have shown thatneuroprogenitor cells (NPCs) survive cryopreservation and elicit nearlyidentical properties after a preservation cycle. Therefore, anadditional advantage of the present invention is the multiple uses ofthe same NPC stock over long periods of time.

In addition, the BBB model of the present invention can be manipulatedto consist of larger populations of neuroprogenitor cells (NPCs) ratherthan mature cell types to represent more of the immature BBB found inthe embryo or to yield an improved model in vitro system for the studyof NPC-endothelial interactions. Such interactions may ultimatelydictate the success of stem cell therapies given that neuroprogenitorcells (NPCs) lie in the vascular niche in the adult brain.

In summary, using neuroprogenitor cells (NPCs) as source tissue has thepotential to revolutionize the in vitro BBB market by providing ease ofuse, reliability, and the most realistic in vivo-like properties todate.

Method of Creating a Model BBB

In one embodiment, the present invention is a method of creating amulticellular blood-brain barrier model, preferably using primary brainmicrovascular endothelial cells and neural progenitor cells as sourcetissues. One would typically begin by creating a culture ofpuromycin-purified primary brain microvascular endothelial cells(BMECs). In general, a suitable preparation is any monolayer of BMECthat is quite pure and possesses well developed tight junctions as wellas expressing endothelial markers such as von Willebrand factor,PECAM-1, and p-glycoprotein. The Examples disclose the isolation of ratbrain capillaries and the plating of the capillary cells on a permeablesupport. Although rat BMECs are specifically used in the Examples, oneof skill in the art would understand that one could substitute othermammalian endothelial cells, most specifically other rodent cells andprimate cells, including human cells using appropriate isolation andculture techniques (reviewed in Deli et al., 2005).

The BMEC monolayer is suitable for initiating a co-culture when thecells are confluent and express a TEER of 20-50, preferably greater than35 Ohm×cm². This permeability measurement indicates an impermeability toion diffusion characteristic of a confluent monolayer in the absence ofco-culture. At this stage, BMECs express their typical spindle shapedmorphology and form a monolayer without defects.

The permeable support is preferably situated in a commercially availableTRANSWELL™ filter setup (or similar) that consists of an upper and lowerchamber separated by a permeable membrane. FIG. 1 demonstrates aschematic version of a suitable apparatus.

In one version of the invention, cortical neural progenitor cells (NPCs)are isolated (preferably as described in the Examples) and disassociatedinto a single cell suspension. (Although rat neuroprogenitor cells(NPCs) are specifically used in the Examples, one of skill in the artwould understand that one could substitute other mammalian cells, mostspecifically other rodent cells and primate cells, including humancells.) The cells are seeded, preferably at a density of 2×10⁵ cells/mlin NPC culture medium along with epidermal growth factor, fibroblastgrowth factor and heparin (see Weidenfeller et al. 2007 and/or Example Ifor detailed preferred culture medium and approaches).

In one embodiment of the invention, cells are first grown asfree-floating neurospheres and then co-cultured with the BMECs,preferably as described in the Examples.

A preferable co-culture of BMECs and neuroprogenitor cells (NPCs) isdescribed at more length in the Examples. In brief, the neurospheres arepreferably collected in vitro four days after isolation (DIV 4) andenzymatically treated. The cells are then counted and plated in a lowercompartment of the TRANSWELL™ filter system with medium allowing NPCdifferentiation. Although the Example shows cells plated at the bottomof the lower compartment of the TRANSWELL™ filter system, theneuroprogenitor cells (NPCs) could also be grown in direct contact withthe BMECs. This could be by, for example, growing the neuroprogenitorcells (NPCs) on the reverse side of the TRANSWELL™ filter itself.

In another embodiment of the present invention, the neuroprogenitorcells (NPCs) are pre-differentiated before combination with the BMECs.By 8 days the pre-differentiated cells no longer display the expressionof nestin, the NPC marker. Populations of cells expressing GFAP(astrocytes), β-tubulin (neurons), and myelin basic protein(oligodendrocytes) are present. Example II is an example of anembodiment of the present invention in which the neuroprogenitor cells(NPCs) are pre-differentiated for 6-14 days. While twelve days is apreferred pre-differentiation time, Applicants note that the presentinvention is suitable for neuroprogenitor cells (NPCs) that arepre-differentiated, preferably between 6 and 14 days or under conditionsthat allow substantial differentiation into astrocyte and neuronalmixtures to occur.

After co-culture of BMECs and neuroprogenitor cells (NPCs), one may wishto obtain a TEER measurement of the resulting BBB. Preferably, the TEERwill be greater than 100 Ohm×cm². The Examples below show TEERmeasurements at 100-120 Ohm×cm².

We envision that one may wish to pre-differentiate the neuroprogenitorcells (NPCs) for a varied number of days and under various conditionsdepending on the in vitro model needed. Early on afterpredifferentiation there are still nestin-positive cells in this invitro system resulting in a four cell system together with theastrocytes, neurons, and BMECs as a model for early development withearly induction properties. When pre-differentiated for a longer time(for example, 8-14 days for the rat), nestin-positive cells fullydifferentiate into astrocytes and neurons resulting in a more adult-likein vitro system capable of maintaining high TEER and improved BMECspermeability properties for a longer period of time. In addition, asdescribed earlier, various cofactors (bone morphogenic protein, retinoicacid, cyclic AMP) can be added to the differentiation medium to helptune the ratios of the different cell types. After one has co-culturedBMECs and pre-differentiated neuroprogenitor cells (NPCs), one may wishto take a TEER measurement. Preferably, this measurement will be over100 Ohm×cm². Applicants envision that one will obtain TEERs of 150, 200,or preferably 250 Ohm×cm² upon optimization using factors describedabove and below in this specification.

The puromycin purification step is important (but not required). One ofskill in the art would understand that puromycin may be substituted byother toxins such as vincristine, vinblastine, colchicine that arerecognized by the efflux transport system of the brain endothelialcells.

In one embodiment of the invention, the method is performed with primaryrat BMECs and embryonic rat cortical neuroprogenitor cells (NPCs).However, one of skill in the art would understand that other mammaliancell species are substitutable. Additionally, one may wish to create ahybrid barrier with rat/human or human/non-human primate components.

Human nestin-positive cells would be treated similarly to the treatmentdescribed for rat nestin-positive cells in the Examples. An importantexception is that human neurospheres are passaged every 14 days bysectioning into 200-μm sections prior to seeding into fresh growthmedium. The isolation and culture of human endothelial cells would beperformed similarly to that described for rat BMECs, using instead humanautopsy brain tissue from which the capillaries are isolated.

For cultures based on species other than rat and human, the cultureconditions (growth factors and time in culture) have to be adjustedaccording to the best induction of BBB properties (expression ofspecific marker proteins, high TEER and low permeability).

In another embodiment of the invention, one would substitute the primaryBMECs described above with embryonic stem cell-derived endothelial cells(ECs). ECs can be derived from embryonic stem cells with reasonably highyield and purity (Kubo et al., 2005; Kaufman et al., U.S. Pat. No.7,176,023) and would provide an unlimited supply of ECs that would avoidthe isolation of adult BMECs. This would clearly be an advantage for ahuman BBB model, where reliance on autopsy tissue would be removed. Theembryonic ECs that are essentially naïve in that they have not beenexposed to the brain microenvironment would be cultured with eitherneuroprogenitor cells (NPCs) or pre-differentiated neuroprogenitor cells(NPCs) to induce BBB properties and complete the in vitro BBB model. Onefamiliar with the art would also understand that adult NPC could be usedin replacement of embryonic NPC.

BBB Model

In another embodiment, the present invention is an in vitro blood-brainbarrier model comprising three components within a liquid-containingvessel. The first component is a confluent layer of brain microvascularendothelial cells, as described above. The second component is apermeable membrane support, preferably the TRANSWELL™ permeable filtersupport (pore size 0.4 μm) described in the exhibits. For permeabilitystudies with higher molecular weight components (antibodies, phage), thepore sizes can be adjusted (1-3 μm filters are available). Also suitablewould be hollow fibers, side-by-side chambers, and different poredensity or pore size filers. The third component comprises eitherpre-differentiated neural progenitor cells or undifferentiated embryonicneural progenitor cells that are co-cultured with BMECs. Afterco-culture, one could remove the NPC or NPC-derived cells and retaingood BBB characteristics for a period of time. See FIG. 1 for apreferable model. The first and second components are a permeablebarrier between a top and bottom section of the vessel. Typically, thevessel is filled with culture medium as described in Examples I-III (NPCculture medium [DMEM:HAMS-F12 at 3:1 supplemented with B27 (2% v/v),epidermal growth factor (EGF, 20 ng/mL) fibroblast growth factor (FGF-2,20 ng/ML), and heparin (5 μg/mL)].

The embryonic neural progenitor cells that are co-cultured with theBMECs having the characteristic cellular distributions presented, (FIG.4) will yield a BBB model having early inductive characteristics.Pre-differentiated embryonic neural progenitor cells could have avariety of cellular distributions for astrocytes, neurons, andoligodendrocytes that will all yield improved BBB model characteristics.The methods described in Example II, FIG. 8 yield one such combinationthat outperforms simple astrocyte culture.

In one embodiment, one may construct the model described above andremove the neural cells after a TEER of greater than 100 Ohm×cm² hasbeen obtained.

Additionally, a suitable BBB has particular permeability characteristicsthat are useful in a BBB model. The Examples demonstrate specificcharacteristics. For example, Example I describes the effects ofneuroprogenitor cells (NPCs) on BMEC morphology and improved tightjunction fidelity. In addition, Examples describe the improvement inimpermeability by TEER measurement. The TEER measurements are often inthe range of 100-120 Ohm×cm². This range allows significant improvementin small molecule permeability measurements and is also regarded as asuitable permeability range by researchers experienced in the art.Similarly, Example II indicates the improved permeability of BBB modelsusing pre-differentiated neural progenitor cells that also performsbetter than astrocyte co-culture, the current state-of-the-art.Applicants envision that with optimization, TEER measurements usingpre-differentiated neural progenitor cells will improve to 150 Ohm×cm²,200 Ohm×cm² or most preferably 250 Ohm×cm².

In another embodiment of the invention, the first component comprisesdifferentiated embryonic stem cells that have been differentiated intoendothelial cells. A suitable BBB model will have characteristicsdescribed above for the primary BMEC derived blood-brain barrier.

One may also wish to manipulate the components of the BBB model tocreate a membrane that is more suitable for an individual study. Forexample, one may wish to manipulate the cell population so that themodel will more clearly mimic the BBB from different parts of the brain.Using neuroprogenitor cells (NPCs) derived from different regions of thebrain can result in different astrocyte-neuron ratios (Ostenfeld et al.,2002) and the resultant brain cells will likely have regionalspecificity in terms of their BBB-inductive properties. Also, asdescribed earlier, the use of undifferentiated versus pre-differentiatedneuroprogenitor cells (NPCs) can be used to tune the BBB model forspecific applications ranging from embryonic drug delivery to stem celltransplantation. As a further example, using different substrates forboth the BMECs and neural progenitor cell components (different poresizes and extracellular matrix coatings) or even growing the BMECs andneural progenitor cells in direct contact to form capillary likestructures can lead to BBB models that can be used to investigate avariety of pharmacologic, toxicologic, and developmental phenomena.Monitoring characteristics that are critical for representing the invivo situation (high TEER, expression and polarization of transporterslike the p-glycoprotein drug efflux transporter and the GLUT1 glucosetransporter, expression and maintenance of well-developed tightjunctions) would help determine the appropriate approach for aparticular application.

Use of the Model BBB to Analyze Compounds.

One would wish to use the model BBB of the present invention to analyzepermeability characteristics of various test compounds. Most preferablyone would analyze the compound in the following manner:

Many methods can be used to determine BBB permeability as well as uptakeand efflux rates using an in vitro model. As an example, the rates fortrans-BBB transport can be used to directly determine a pseudosteady-state permeability value (Pe) which is an estimate of in vivopermeability for a particular pharmaceutical compound, protein, or drugcarrier. With the assumption that the concentration in the upper fluidcompartment is static, and with a correction for the resistance providedby the membrane itself, the permeability can be readily determined foreach test compound. Briefly, the transcytosis rate (Q) can be divided bythe concentration of material added to the apical chamber (Ca) and thearea of the membrane (Am) to yield a total permeability (Pt) thatincludes resistances due to the monolayer and the membrane (Pt=Q/[AmCa],or using clearance volume terminology Pt is equal to the slope of theclearance volume versus time line divided by the membrane area).Subsequently, Pe can be calculated by correcting for the resistancesupplied by the cell free membrane (Pm) (1/Pe=1/Pt−1/Pm) (Bickel, U. Howto measure drug transport across the blood-brain barrier. NeuroRx 2005,2, 15-26.). This strategy was used successfully to determine thetranscytosis rate of fluorescein and its associated permeability(Pe=3.3×10-4 cm/min) through the in vitro model as demonstrated inExample I.

EXAMPLES Example I Differentiating Embryonic Neural Progenitor CellsInduce Blood-Brain Barrier Properties Materials and Methods

Isolation of Rat Brain Microvessel Endothelial Cells

The isolation of rat brain capillaries was performed as previouslydescribed (Weidenfeller et al. 2005; Calabria et al. 2006). Briefly, themeninges-free cortices from adult male Sprague Dawley rats (220-250 g)were mashed with forceps, and thoroughly triturated. Capillaries wereseparated from surrounding tissue by sequential digestion/densitycentrifugation steps with type 2 collagenase (Worthington BiochemicalCorporation) and Collagenase/dispase (Roche Applied Science). Thecapillaries were plated onto 1.12 cm² TRANSWELL-CLEAR™ filter permeablesupports (0.4 μm pore size, Corning) coated with collagen IV/fibronectinin puromycin-supplemented medium (4 μg/mL) containing DMEM, 20% bovineplatelet poor plasma derived serum, 1 ng/mL human basic fibroblastgrowth factor (FGF-2/bFGF, R&D Systems), 1 μg/mL heparin, 2 mML-glutamine, and an antibiotic-antimycotic solution(Penicillin-Streptomycin-Amphotericin (PSA): 100 U/mL penicillin, 100μg/mL streptomycin, and 0.25 μg/mL amphotericin). Cultures weremaintained in a 37° C. incubator under humidified 5% CO₂/95% air.

Isolation and Culture of Rat Cortical Embryonic Neural Progenitor Cells

Rat cortical NPC were isolated as described previously (Ostenfeld et al.2002). The cortices were dissected from E14 rat brains. The tissue wastreated with Accutase™ (Innovative Cell Technologies, San Diego, Calif.,USA) for 10 minutes at 37° C., washed twice in DMEM, and thendissociated into a single cell suspension. Cells were initially seededat a density of 2×10⁵ cells/ml in a T25 flask in defined serum-free NPCculture medium [DMEM:HAMS-F12 at 3:1 supplemented with B27 (2% v/v),epidermal growth factor (EGF, 20 ng/ml), fibroblast growth factor(FGF-2, 20 ng/ml), and heparin (5 μg/ml)]. Cells were grown asfree-floating neurospheres for 3 days and then used for co-culture withBMEC on day in vitro 4 (DIV4).

Isolation of Astrocytes

Cortices from neonatal rats (P6) were minced in a petridish containingice-cold Hanks' Balanced Salt Solution (HBSS). The minced tissue wascentrifuged for 2 min (500 g), resuspended in HBSS containing trypsin(0.5 mg/ml) and incubated at 37° C. for 25 min in a shaker bath. Thetrypsinized tissue was triturated and the cell suspension was filteredthrough a 70 μm mesh. 3×10⁴ cells/cm² were plated in DMEM containing 10%FBS, 10% horse serum, 2 mM L-glutamine, and PSA. Medium was changedevery third day and cells were treated with 0.25 mM dibutyryl cAMP for 3days prior to co-culture with BMEC to induce an in vivo-like phenotype(Segovia et al. 1994; Enkvist et al. 1996). The presence ofGFAP-expressing astrocytes was confirmed by immunocytochemistry.

Co-Culture of BMEC with NPC or Astrocytes

Neurospheres were collected on DIV4, treated with ACCUTASE™, and washedtwice in DMEM. Live cells were counted on a hemacytometer, and 2.5×10⁵NPC/cm² were plated in the lower compartment in either mitogen-freemedium allowing NPC differentiation (DMEM:HAMS-F12 at 3:1, 2% v/v B27,1% FBS, and PSA, with poly L-lysine/laminin coating) or withmitogen-containing medium to suppress differentiation (mitogen-freemedium plus 20 ng/mL EGF and 10 ng/mL FGF-2, without polyL-lysine/laminin coating) (Ostenfeld et al. 2002). The TRANSWELL™ filtercontaining the confluent BMEC was then added to complete the co-culturesystem (FIG. 1). Mitogen-mediated suppression of NPC differentiation inthe presence or absence of BMEC was confirmed by anti-nestin, anti-GFAPand anti-βIII tubulin staining. In this way, it was determined that theNPC populations were GFAP and βIII tubulin negative prior to and afterBMEC co-culture in mitogen-containing medium. NPC densities up to 1×10⁶NPC/cm² were tested, but were not found to yield any increases in TEERinduction above that seen with the 2.5×10⁵ NPC/cm² plating density.Embryonic mouse fibroblasts (3T3, ATCC) were used as a non-neuralco-culture control.

One day prior to co-culture, astrocytes were preconditioned inmitogen-free medium to avoid effects that serum withdrawal could have onthe temporal response of astrocyte induction. After 24 hours ofpreconditioning, astrocytes were treated with trypsin-EDTA solution andsingle cells were resuspended in mitogen-free medium. A total of6.25×10⁴ astrocytes/cm² were added to the lower compartment inmitogen-free medium, and the filter insert with the confluent BMECmonolayer was added.

Immunocytochemistry

All steps were performed at 20° C. The BMEC and NPC cultures were gentlywashed three times with 0.01 M PBS and fixed with paraformaldehyde (4%w/v in PBS). After blocking and permeabilization (10% goat serumcontaining 0.1% Triton X-100 in PBS (PBSG), 30 min), primary antibodies(anti-nestin, [BD Biosciences], rabbit anti-glial fibrillary acidicprotein [GFAP, DAKO Cytomation], anti-βIII tubulin [BD Biosciences],anti-von Willebrand factor [Sigma], anti-occludin, anti-zonulaoccluden-1, mouse anti-claudin 5 [Invitrogen], primary antibody mix forMBP and CNPase detection [Orion Biosolutions]) were diluted in PBSG with3% goat serum and incubated with samples for 1 h. Samples were washedwith PBS and incubated with secondary antibodies (Texas Red goatanti-rabbit IgG, Alexa Fluor goat anti-mouse IgG antibody) diluted inPBSG with 3% goat serum for 1 h. DAPI nuclear stain at a concentrationof 300 nM in PBS was added to the wells for 5 min. Forimmunocytochemistry of brain tissue sections, freshly isolated E14 ratbrains were embedded in tissue freezing medium, snap frozen in liquidnitrogen, sectioned, and labeled as described above.

Resistance Measurements

Transendothelial electrical resistance (TEER) was measured using an EVOMvoltohmmeter (World Precision Instruments). Resistance values (Ω×cm²)were corrected by subtracting the resistance of a substrate coated,empty filter. At each time point, three TEER measurements were taken perTRANSWELL-CLEAR™ filter to yield an average TEER value for each filter.Subsequently, TEER values for triplicate filters at each culturecondition were used to compute the mean and standard deviationsreported.

Permeability Studies

The permeability was assessed by determining the flux of fluoresceinthrough the BMEC monolayer. Fluorescein sodium salt in DMEM was added tothe apical filter compartment to produce a uniform initial concentrationof 1 μM. Subsequently, 200 μl were removed from the basolateralcompartment after 0, 15, 30, 45, and 60 min. The fluorescence wasmeasured with the FLx800 fluorescence reader (Bio-Tek Instruments) andthe rates of fluorescein accumulation in the lower compartment used todetermine the permeability as described previously (Perriere et al.2005).

Quantitative Analysis of Cultures

Counting of NPC-derived cell types was performed by overlay ofanti-nestin (undifferentiated NPC), anti-13111 tubulin (neurons), andDAPI images or by overlay of anti-nestin, anti-GFAP (astrocytes), andDAPI images. The cell distribution was assessed by determining thepercentage of cells positively-labeled for a particular marker. For thisdetermination, 5 random fields for each type of labeling (βIIItubulin/Nestin or GFAP/Nestin, ˜1000 total cells each condition) werecounted at a magnification of 40×. Proliferation of BMEC was evaluatedby BrdU incorporation with the 5-Bromo-2″-deoxy-uridine Labeling andDetection Kit 1 (Roche Applied Science) according to the manufacturer'sinstructions. BrdU was added to the cultures at the beginning of the 24h co-culture of BMEC with NPC. The cells were fixed after 24 h, totalBMEC numbers were assessed by DAPI nuclear stain, and the percentage ofBMEC incorporating BrdU was determined for 6 different fields on each of3 filters (800 cells for each condition). An analogous procedure wasused to assess NPC BrdU incorporation and total cell numbers of NPC. Thepercentage of BMEC containing frayed junctions over a significantfraction (greater than 10%) of their total cell border was determined byrandomly choosing microscope fields in phase contrast mode wherejunctional ultrastructure is not visible. The immunocytochemical imagesfor occludin labeling were then acquired in fluorescence mode. Junctionsbetween adjacent cells (100 cells per image with 5 images total for eachcondition) were defined as frayed if immunolabeling illuminated tightjunction protrusions that are not parallel to the cell-cell border.

Results

Primary Brain Endothelial Cell-Embryonic NPC Co-Culture Model

The influence of NPC on the barrier properties of adult BMEC wasinvestigated using a novel in vitro model consisting of primary rat BMECand embryonic rat cortical NPC. Since BMEC isolated from adult brainsde-differentiate in vitro (Krizbai and Deli 2003), they have been widelyused to study BBB induction and modulation, although they still possesssome level of BBB properties. NPC and BMEC were co-cultured togetherusing a microporous filter setup (TRANSWELL-CLEAR™ filter) with an uppercompartment and a lower compartment representing the blood and brainside of the blood-brain barrier (BBB), respectively (FIG. 1). The filtersetup allows the in situ measurement of the transendothelial electricalresistance (TEER) yielding information regarding the integrity of theBMEC monolayer by monitoring the paracellular flux of smallelectrolytes. Puromycin-purified BMEC were cultured on the upper surfaceof the filter membrane, and after 3 days in vitro (DIV3) had grown toconfluence as determined by phase contrast microscopy. Puromycintreatment ensured a nearly 100% pure endothelial monolayer, and we andothers have demonstrated that this rodent in vitro BBB model displayswell defined tight junctions and forms an impermeable barrier to smallmolecule tracers (Perriere et al. 2005; Weidenfeller et al. 2005;Calabria et al. 2006). Importantly, the model has also proven reliablefor the measurement of BMEC response to inductive factors such as cAMP,astrocytes, and glucocorticoids (Perriere et al. 2005; Weidenfeller etal. 2005; Calabria et al. 2006), and was therefore suitable for thetesting of NPC inductive capacity. In order to co-culture NPC with theBMEC monolayer, freshly isolated NPC from E14 rats were expanded for 3days in EGF and FGF-2 mitogen-containing medium (see Materials andMethods for details) and then cultured in the lower compartment suchthat the BMEC and NPC could interact via soluble mediators. Whenco-cultured in mitogen-free medium, NPC attached to the polyL-lysine/laminin coated substrate in the lower compartment, generating amixture of NPC (nestin-positive) and differentiated progeny includingastrocytes (GFAP-positive) and neurons (βIII tubulin-positive).Co-culture in the presence of mitogens supported solely proliferation asnestin-positive NPC.

NPC Influence on BMEC TEER and Permeability

The in vitro co-culture model was used to investigate a possibleinvolvement of NPC in the induction of BBB properties in the BMECmonolayer. In order to determine whether NPC or NPC-derived cells couldaffect the in vitro barrier phenotype of BMEC monolayers, the TEER wasmeasured in the presence or absence of differentiating NPC (mitogen-freeconditions). TEER measurements after 24 hours of co-culture indicated a47% increase in monolayer TEER with NPC (110±5 Ω×cm²) when compared tocontrol BMEC cultures lacking NPC (75±4 Ω×cm²), indicating an earlyinductive response to soluble factors released by NPC (FIG. 2, columns 1and 2). The barrier-enhancing effect in the presence of differentiatingNPC was also observed in permeability studies where NPC co-culturereduced the monolayer permeability for fluorescein sodium salt by 33%with NPC influences (Table 1). This decrease in diffusion of theBBB-impermeable fluorescein directly correlated with the increase inTEER. In addition, to support the presence of soluble factors thatmediate the increases in TEER, the medium conditioned by 24 hours ofco-culture was serially applied to fresh BMEC monolayers. The BMEC-NPCco-culture conditioned medium again induced BMEC TEER (150±6%) comparedwith BMEC monolayers grown in medium conditioned by BMEC alone. Finally,even when NPC were maintained in an undifferentiated state for longerperiods of 1-5 weeks in vitro prior to co-culture with BMEC, theinduction properties were still observed under the mitogen-freeconditions (31-43% increases in TEER).

In contrast, conditioned medium (24 h) from NPC differentiating in theabsence of BMEC did not show an effect on the TEER within 24 h afterapplication to BMEC (FIG. 2, column 4). When co-cultured with NPC thatwere pre-differentiated for 24 hours in the absence of BMEC, TEERincreases were attenuated (11±6%) (FIG. 2, column 3). In order todetermine whether proliferating, undifferentiated nestin-positive NPC orconditioned medium from proliferating, undifferentiated NPC can inducean increase in TEER, BMEC were co-cultured with NPC in the presence ofmitogens or with NPC culture-conditioned medium from 24 hourmitogen-treated, proliferating NPC, respectively (FIG. 2, columns 5 and6). No induction in BMEC TEER could be observed under these conditionsafter 24 h. Finally, the effects of non-neural embryonic 3T3 fibroblastson BMEC were investigated and like the mitogen-treated NPC, no inductionwas observed (data not shown). The lack of induction in the presence ofundifferentiated, proliferating NPC or 3T3 fibroblasts indicated thatthe simple presence of another cell type was not responsible for changesin phenotype observed in the presence of differentiating NPC.

Influence of NPC Co-Culture on BMEC Morphology

The tight junctions of BMEC cultured alone or co-cultured with NPC wereinvestigated to determine if NPC were capable of influencing BMECcell-cell contacts or cell morphology in a way that could account forthe increased TEER and decreased fluorescein permeability. BMEC wereprobed with antibodies against tight junction proteins ZO-1, occludin,and claudin 5 in the presence (FIG. 3 A, C, E) or absence (FIG. 3. B, D,F) of differentiating NPC. In the presence of NPC the majority of theBMEC show well-established tight junctions and a distinct occludin,claudin 5, and ZO-1 staining. In the absence of NPC, cell-cell junctionswere also evident, but tight junction staining indicated an irregular,frayed staining pattern in 65% of the BMEC while in the presence of NPC,only 34% of the BMEC exhibited such a junctional structure (Table 1,FIG. 3). The cell morphology and cell size were indistinguishable underthese two conditions (Table 1). BMEC with or without NPC co-culture wereprobed for F-actin localization, and no difference between BMEC-NPCco-culture and BMEC monocultures was detected. In both cases, a strongperi-junctional actin localization together with intracellular actinfilaments was observed (data not shown).

NPC Effects on BMEC Proliferation

Next, the filter density of BMEC was evaluated to determine whether ornot the increased TEER values were the result of a tighter monolayerpacking. BMEC monolayers with or without NPC co-culture were assessedusing DAPI nuclear staining and 5-Bromo-2″-deoxy-uridine (BrdU)incorporation to investigate BMEC density and proliferation,respectively. As Table 1 indicates, no significant difference in thenumber of proliferating endothelial cells or BMEC cell density wasobserved between the NPC-BMEC co-cultures and BMEC mono-cultures.

Potential Inductive Cell Types in the Co-Culture System

In order to determine the NPC-derived cell types that might beresponsible for the induction of TEER and the observed changes injunctional structure, NPC progeny in the basolateral compartment wereprobed for astrocytic (GFAP), neuronal (βIII-tubulin) and progenitorcell (nestin) markers (FIG. 4). There was no significant change in thetotal number of NPC-derived cells in the lower compartment with orwithout BMEC being present, and the number of proliferating BrdU⁺ cellswas also the same (˜40%). The majority of NPC-derived cells remainnestin-positive (65±5.3%) after 24 hours in co-culture with BMEC duringwhich time the NPC induction effects are first observed. Results alsoindicated that NPC differentiate into neuronal and glial cells. Thesecond largest cell population was positive for both GFAP and nestin(15.6±3.8%) indicating that these cells are likely immature astrocytes.A small population (9.7±2.7%) of cells was solely GFAP-positive andconsidered to be more mature astrocytes. Very few neurons (6.5%±0.7%)and immature βIII tubulin- and nestin-positive neurons (3.3±1.0%) weregenerated in the presence of BMEC in the 24-hour timeframe. In theabsence of BMEC, more NPC differentiated towards a neuronal fate whilethe numbers of solely GFAP-positive and GFAP/nestin co-stained cellsremained unaffected. The increased number of neurons was accompanied bya lower number of nestin-positive cells in the absence of BMEC(46±4.2%), indicating a higher propensity for NPC to differentiatewithout BMEC influences. Oligodendrocytes were not detectable byimmunofluorescence using antibodies against myelin basic protein or2′3′-cyclic-nucleotide-3′ phosphodiesterase (CNPase).

Effects of BMEC on NPC Morphology

Further evidence of BMEC-NPC crosstalk was gathered by investigating theinfluence of BMEC on the morphology of NPC-derived cell types.Co-culturing with primary BMEC significantly influences the morphologyof the neural progenitor cells. NPC that were allowed to proliferate anddifferentiate (mitogen-free conditions) in the absence of endothelialcells possessed small cell bodies and displayed multiple thin processestypical of maturing astrocytes and neurons (FIGS. 5 A and C). Incontrast, the number and length of processes is reduced in the presenceof BMEC and the cells have a much more flattened precursor-likemorphology (FIGS. 5 B and D). This effect was detectable for each of theastrocyte, neuron, and NPC cell types.

Determination of Blood Vessel-NPC Localization in E14 Rat Brain

In order to correlate the in vitro results with the actual cellulardistribution observed in vivo in the developing E14 rat cortex, thedistributions of NPC, astrocytes, and neurons were investigated. NPC, asdetermined by nestin expression, could be identified throughout thewhole cortex with a high density at the inner cortex close to theventricle (FIG. 6 A, B, D). Radial glia spanned to the pial surface andwere identified as elongated nestin-positive cells. In the pial-proximalarea, a high density of neurons can also be observed (FIG. 6 B, C).Blood vessels could be easily identified throughout the embryonic brainby anti-Glut1, anti-PECAM1 and anti-von Willebrand factor labeling, andthey were found in regions with high numbers of NPC and neurons (FIG. 6A). In contrast, astrocytes were not detectable in the E14 rat cortex(FIG. 6 D). Finally, the tight junction protein occludin was notdetectable indicating the presence of immature endothelial junctions inthe E14 rat brain (FIG. 6 C).

Comparison of Barrier Induction Mediated by Postnatal Astrocytes VersusEmbryonic NPC

Although the majority of the NPC-derived cells in the BMEC-NPCco-cultures were nestin positive, small percentages of astrocytes (GFAP)or immature astrocytes (GFAP/nestin) were present. Thus, in an effort todistinguish between the effects commonly associated with astrocytes andthose mediated by dividing and differentiating NPC, the TEER inductionproperties of these two situations were directly compared. In parallelto NPC-BMEC co-culture, BMEC were also co-cultured with postnatal (P6)astrocytes at a density corresponding to the number of GFAP-positivecells (25%) counted in the BMEC-NPC co-culture experiment. Based onthese counts, BMEC were co-cultured either with astrocytes(6.25×10⁴/cm²) or NPC (2.5×10⁵/cm²), and the TEER was monitored as afunction of time (FIG. 7). The data indicate that NPC induced a 24%increase in BMEC TEER (96±5 Ω×cm²) compared with the untreated controlafter just 24 hours (77±5 Ω×cm²), while astrocytes did not induce astatistically significant increase in TEER (78±4 Ω×cm²) during that timeperiod. The maximum TEER reached by the astrocyte co-culture was similarto that achieved by NPC and was detected after 48 hours in co-culturewhere astrocytes (97±5 Ω×cm²) result in a 38% increase in TEER. By 72hours, the TEER in NPC co-culture decreased to the level of themonoculture control. However, in the case of astrocyte co-culture, theTEER remained elevated out to 120 hours. To investigate whether thelower overall density of astrocytes compared with NPC contributed to thedelay in inductive response, co-culture of BMEC was also performed witha confluent monolayer of astrocytes. The higher density astrocyteco-culture yielded the same magnitude of TEER induction and the sametemporal response as that seen for the low-density astrocyte co-culture,including the delay in TEER induction (data not shown).

Discussion

In this study, the influences of NPC on the BBB properties of BMEC wereinvestigated. An in vitro model consisting of primary rat BMEC andembryonic NPC was evaluated for its barrier properties and compared witha BMEC model lacking NPC. NPC significantly influenced BMECs by inducingTEER, reducing permeability, and affecting tight junction structure.Barrier-inducing effects were only observed in the presence ofdifferentiating NPC, while proliferating NPC in the presence of mitogensyielded no influence on BMEC monolayer TEER. Finally,barrier-strengthening effects elicited by NPC were distinguishable fromastrocytic induction in terms of both the timing and duration of TEERinduction. To our knowledge, this is the first demonstration of thedirect influence of NPC on BBB properties of BMEC.

The increase in BMEC TEER was detectable after 24 hours of co-culturewith NPC in mitogen-free medium and correlated with a decrease influorescein permeability. Since fluorescein is a small molecule thatdoes not appreciably cross the BBB in vivo (Hoffman and Olszewski 1961),these measurements serve as a barometer for the functionalimpermeability of BBB models. The absolute TEER values (70-120 Ω×cm²)achieved in this study were typical of TEER values reported for otherrat and mouse BBB models (de Vries et al. 1996; Perriere et al. 2005;Weidenfeller et al. 2005; Calabria et al. 2006; K is et al. 2001). Also,as a comparison, the effect of NPC on the BMEC fluorescein permeability(3.3±0.5×10⁻⁴ cm/min, 33% permeability reduction) was of a similarmagnitude to that previously observed upon co-culturing rat BMEC withastrocytes (2.7×10⁻⁴ cm/min, (K is et al. 2001; Perriere et al. 2005)).It was possible that the observed effects resulted from a higher BMECdensity on the filter membrane. However, NPC did not influence theendothelial cell density and did not yield higher numbers ofproliferating BMEC. Therefore, it was concluded that NPC induction ofBMEC properties was not simply based on a more tightly packed monolayer,nor was it a result of newly formed BMEC having optimized propertiesbecause they were generated in the presence of NPC influences. Instead,the effect correlated with tight junction fidelity as a large fractionof the BMEC possess junctions that are continuous in the presence of NPC(33.5% frayed, 110 Ω×cm²), while in the absence of NPC cell-celljunctions are predominantly frayed (65.1% frayed, 75 Ω×cm²). Thedecrease in frayed BMEC tight junctions has also been previously notedto correlate with higher TEER and lower permeability in BMEC cultures(Weidenfeller et al. 2005; Calabria et al. 2006). Similar to the casewith NPC induction, treatment with BBB-inducing glucocorticoids such ascorticosterone (21% frayed, 168 Ω×cm²) or hydrocortisone (12% frayed,218 Ω×cm²) decreases the number of frayed junctions and increases theTEER while also lowering the fluorescein permeability (0.66×10⁻⁴ cm/minfor hydrocortisone) (Weidenfeller et al. 2005; Calabria et al. 2006).Taken together, these results suggest that the improved barrierproperties in the presence of NPC are likely a result of improvedcell-cell junctional contacts.

Since the BMEC and NPC were separated by a microporous filter membraneand 1 mm of culture medium (FIG. 1), the barrier induction was clearlymediated by soluble factors. The induction was observed in mitogen-freemedium as NPC begin differentiating into astrocytes and neurons, but itwas not detectable in the presence of mitogens that keep NPC in anundifferentiated, nestin-positive state (Gage 2000; Ostenfeld et al.2002). This finding suggests that some component of the differentiationprocess is likely required for BMEC barrier induction and that NPCproliferating in an undifferentiated state do not have a majorinfluence. Although the presence of mitogens themselves might haveinfluenced the integrity of the BMEC monolayer thereby masking theeffects of factors released by proliferating, nestin-positive NPC (Sobueet al. 1999), this was not evident from TEER measurements of controlBMEC in mitogen-containing medium. The lack of BMEC induction in thepresence of 3T3 fibroblasts, proliferating nestin-positive NPC orpostnatal astrocytes at 24 hours also indicates that the observedinduction with differentiating NPC is not a generic trophic response dueto the presence of another proliferating cell type, but instead showsthat the properties specific to differentiating NPC are required. WhenBMEC were cultured in medium that was conditioned by differentiating NPCfor 24 h, no TEER induction was detected. Therefore, the BMEC presenceduring the NPC differentiation process was required for the NPC torelease BBB-inducing soluble factors, and this finding implicates abidirectional communication between BMEC and NPC. The requisiteinteraction between NPC and BMEC was further validated by demonstratingthe capacity of co-culture conditioned medium to increase the TEER whenserially applied to fresh BMEC monolayers. In order to determine ifpre-differentiated NPC lacking early BMEC influences release inductivefactors, NPC were pre-differentiated in the absence of mitogens andsubsequently added to the BMEC monolayer. After 24 h in co-culture, onlya slight increase in BMEC TEER was detectable (11% pre-differentiatedversus 47% co-differentiating NPC). Subsequent to 24 hours ofpre-differentiation in the absence of BMEC, less than 50% of the NPC areundifferentiated (nestin-positive only), likely weakening the effectthat differentiating cells can have compared with a situation whereco-culture is started with a highly pure population of undifferentiatedNPC. These observations support the conclusion that differentiating NPC,rather than differentiated (tubulin- or GFAP-positive) or proliferating(nestin-positive) NPC, stimulate BMEC barrier induction.

NPC proliferate as nestin-positive cells and differentiate into neurons,astrocytes, and oligodendrocytes in mitogen-free conditions (Ostenfeldet al. 2002; Ostenfeld and Svendsen 2004). Thus, NPC-derived astrocytesor neurons could be responsible for the TEER induction. Numerous studieshave demonstrated that astrocytes and neurons have the potential tomodulate BBB tight junctions, transporter expression, and metabolicactivity in vitro and in vivo (Stewart and Wiley 1981; Risau et al.1986b; Savettieri et al. 2000). Very few βIII tubulin-positive neuronsand βIII tubulin/nestin co-positive cells (10% combined) were generatedin the presence of BMEC. Also since more neurons are present when NPCare pre-differentiated prior to co-culture but the resulting inductiveeffect lessened, βIII tubulin-positive neurons do not appear to play asignificant role in the observed induction process. Nearly 16% of theNPC-derived cells were positive for both GFAP and nestin, indicatingthat the second largest population of cells is committed to theastroglial fate but has not yet fully matured. Only 10% were matureastrocytes as defined by GFAP expression. Although astrocytes are stronginducers of BBB properties, the timecourse of TEER induction by NPCindicated that NPC acted earlier while astrocyte effects were moreprolonged. Similarly, the pre-differentiated NPC cultures having 25%GFAP-positive cells also exhibited only weak inductive properties after24 hours (FIG. 2, column 3). Interestingly, the NPC and astrocytescaused the BMEC to reach a similar maximum TEER value indicating acomparable absolute induction capacity of NPC and astrocytes under theseexperimental conditions, although the dynamics of induction clearlydiffered. While this study cannot entirely exclude the possibility thatNPC-derived astrocytes are providing the inductive signals, these datastrongly suggest that differentiating NPC and astrocytes function viadistinct induction mechanisms or at the very least, different temporalprograms.

In addition to the inductive signals provided by NPC, BMEC alsoinfluenced the morphology and differentiation of NPC, furtherimplicating a bidirectional paracrine interaction. The findings of aflattened precursor-like progeny and decreased neuronal production inthe presence of primary BMEC corroborate the results of a previous studythat employed brain endothelial cell lines or pulmonary arteryendothelial cells in embryonic neural stem cell co-cultures (Shen et al.2004). In addition, this previous study also demonstrated that uponremoval of the endothelial cells, neurogenesis was increased (Shen etal. 2004). Other investigations have also implicated endothelialinvolvement in NPC regulation by showing that endothelial cells assistin the recruitment of newly formed neurons (Louissaint et al. 2002) andstimulate astrocyte precursor differentiation into GFAP- andS100β-expressing mature astrocytes (Mi et al. 2001). It also has beensuggested that progenitor contact with microvessels during developmentfavors the astrocyte lineage (Zerlin and Goldman 1997). Finally, NPC arealso found in various regions of the adult brain in close proximity tothe vasculature in the so-called stem cell niche (Doetsch 2003b), In theadult, neurogenesis occurs in foci closely associated with blood vessels(Palmer et al. 2000). There is also evidence implying that angiogenesisand neurogenesis may be co-regulated since they are stimulated by manyof the same factors, such as bFGF, VEGF, IGF-1 and TGFβ. In addition,endothelial cells secrete known neuronal differentiation and survivalfactors (bFGF, IGF-1, VEGF, PDGF, IL8 and BDNF) and a link betweenangiogenesis and neurogenesis is found in the adult songbird brainduring testosterone-induced angiogenesis (Palmer et al. 2000; Jin et al.2002; Louissaint et al. 2002). Thus, bidirectional BMEC-NPCcommunication could play important roles in both embryonic developmentand adult brain plasticity.

The predominant cell types in the developing brain cortex at day E14 areNPC, radial glia, neuroblasts, and neurons (FIG. 6, and references (Basset al. 1992; Saunders et al. 2000; McCarty et al. 2002; Doetsch 2003a)).These cells are found in close proximity to and in contact withdeveloping brain vessels in vivo. The appearance of BBB endothelialproperties in vivo occurs shortly after the blood vessels invade theembryonic brain as endothelial cells begin to thin (Bauer et al. 1993;Stewart and Hayakawa 1994; Bolz et al. 1996), brain vessel permeabilitydecreases and the TEER increases (Risau et al. 1986a; Bauer et al.1995). These early stages of BBB development take place while astrocytesare scarce in the developing brain (FIG. 6, and reference (LeVine andGoldman 1988)). Therefore, it is entirely plausible that brain cellsother than astrocytes may be able to induce early BBB properties inbrain endothelial cells. In the developing brain environment, the NPCdifferentiation process could provide the cues necessary for naïve brainEC to acquire initial BBB properties; whereas, in later stages ofdevelopment, astrocytes would induce further maturation and helpmaintain BBB properties in differentiated EC (FIG. 7). Accordingly, theobservations provided in this study indicate that differentiating NPCmay be important for such early onset of BBB properties in thedeveloping embryonic brain, although additional study will be necessaryto define the exact physiological impact of the reported BMEC-NPCinteractions. Despite the fact that the yields of naïve embryonic brainendothelial cells would be prohibitively low for the study describedhere, it is intriguing to consider using an in vitro model that employsembryonic BMEC or even stem cell-derived EC to provide additionalinsight into the process of BBB and NPC co-development.

Example II Co-Culture of BMEC with Predifferentiated NPC

NPC were isolated from E14 rat brains and expanded for 1 week in FGF2and EGF containing medium as described in Example I (EGF, 20 ng/mL),fibroblast growth factor (FGF-2, 20 ng/mL), and heparin (5 μg/mL).During expansion, half of the medium was replaced with freshmitogen-containing medium every 3 days. After one week, NPC werecryo-preserved as single cells digested with ACCUTASE™ enzyme mixture.After thawing, NPC were allowed to recover for 4 days innitrogen-containing medium, and after formation of spheres, cells weredigested with Accutase™ and live cells were counted on a hemacytometer.2.5×10⁵ NPC/cm² were plated in the lower compartment in mitogen-freemedium allowing NPC differentiation [DMEM:HAMS-F12 at 3:1, 2% v/v B27,Invitrogen Corp. (Carlsbad, Calif.)], and antibiotic-antimycoticsolution (100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mLamphotericin), with poly L-lysine/laminin coating. The differentiationmedium was exchanged every 3 days and after 6-14 days in culture, theTRANSWELL™ filter containing the confluent BMEC was then added tocomplete the co-culture system.

Referring to FIG. 8, NPC that were allowed to differentiate for varioustimes prior to co-culture setup showed different inductive properties inBMEC. 12d-pre-differentiated NPC-derived mixtures from astrocytes andneurons showed the best induction in terms of TEER and performed betterthat postnatal primary astrocyte cultures. Pretreatment of NPC-derivedastrocytes and neurons with cAMP had no influence on the magnitude ofthe TEER induction in BMEC.

Example III Co-Culture of BMEC with Human Co-Differentiating NPC

Human NPC were generally cultured like rat NPC in mitogen-containingmedium. Cells were initially seeded in defined serum-free NPC culturemedium [DMEM:HAMS-F12 at 3:1 supplemented with B27 (2% v/v), epidermalgrowth factor (EGF, 20 ng/mL), fibroblast growth factor (FGF-2, 20ng/mL), and heparin (5 μg/mL)]. During expansion, half of the medium wasreplaced with fresh FGF2 and EGF containing medium every 3 days andspheres were chopped every week. Before inducing the co-differentiationprocess, human NPC were digested with ACCUTASE™ enzyme mixture and livecells were counted on a hemacytometer, and 2.5×10⁵ NPC/cm² were platedin the lower compartment in mitogen-free medium allowing the onset ofNPC differentiation [DMEM:HAMS-F12 at 3:1, 1% FBS, 2% v/v B27,Invitrogen Corp. and antibiotic-antimycotic solution (100 U/mLpenicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin), withpoly L-lysine/laminin coating]. Immediately after plating the NPC inmitogen-free medium, the TRANSWELL™ filter containing the confluent BMECwas then added to complete the co-culture system.

Referring to FIG. 9, human co-differentiating NPC showed a similar timecourse of TEER induction in BMEC as rat co-differentiating NPC. Howeverthe peak of induction is slightly lower in the chimeric system than inthe rat system.

REFERENCES

-   Balabanov R. and Dore-Duffy P. (1998) Role of the CNS microvascular    pericyte in the blood-brain barrier. J Neurosci Res 53, 637-644.-   Bass T., Singer G., Slusser J. and Liuzzi F. J. (1992) Radial glial    interaction with cerebral germinal matrix capillaries in the fetal    baboon. Exp Neurol 118, 126-132.-   Bauer H., Sonnleitner U., Lametschwandtner A., Steiner M., Adam H.    and Bauer H. C. (1995) Ontogenic expression of the erythroid-type    glucose transporter (Glut 1) in the telencephalon of the mouse:    correlation to the tightening of the blood-brain barrier. Brain Res    Dev Brain Res 86, 317-325.-   Bauer H. C. and Bauer H. (2000) Neural induction of the blood-brain    barrier: still an enigma. Cell Mol Neurobiol 20, 13-28.-   Bauer H. C., Bauer H., Lametschwandtner A., Amberger A., Ruiz P. and    Steiner M. (1993) Neovascularization and the appearance of    morphological characteristics of the blood-brain barrier in the    embryonic mouse central nervous system. Brain Res Dev Brain Res 75,    269-278.-   Bolz S., Farrell C. L., Dietz K. and Wolburg H. (1996) Subcellular    distribution of glucose transporter (GLUT-1) during development of    the blood-brain barrier in rats. Cell Tissue Res 284, 355-365.-   Butt A. M., Jones H. C. and Abbott N. J. (1990) Electrical    resistance across the blood-brain barrier in anaesthetized rats: a    developmental study. J Physiol 429, 47-62.-   Calabria A. R., Weidenfeller C., Jones A. R., de Vries H. E. and    Shusta E. V. (2006) Puromycin-purified rat brain microvascular    endothelial cell cultures exhibit improved barrier properties in    response to glucocorticoid induction. J Neurochem 97, 922-933.-   Caley D. W. and Maxwell D. S. (1970) Development of the blood    vessels and extracellular spaces during postnatal maturation of rat    cerebral cortex. J Comp Neurol 138, 31-47.-   de Vries H. E., Blom-Roosemalen M. C., van Oosten M., de Boer A. G.,    van Berkel T. J., Breimer D. D. and Kuiper J. (1996) The influence    of cytokines on the integrity of the blood-brain barrier in vitro. J    Neuroimmunol 64, 37-43.-   Deli, M A, Abráham, C S, Kataoka, Y, and Niwa, M. (2005)    Permeability studies on in vitro blood-brain barrier models:    physiology, pathology, and pharmacology Cell Mol Neurobiol.    (1):59-127.-   Doetsch F. (2003a) The glial identity of neural stem cells. Nat    Neurosci 6, 1127-1134.-   Doetsch F. (2003b) A niche for adult neural stem cells. Curr Opin    Genet Dev 13, 543-550.-   Dziegielewska K. M., Daikuhara Y., Ohnishi T., Waite M. P., Ek J.,    Habgood M. D., Lane M. A., Potter A. and Saunders N. R. (2000)    Fetuin in the developing neocortex of the rat: distribution and    origin. J Comp Neurol 423, 373-388.-   Engelhardt B. (2003) Development of the blood-brain barrier. Cell    Tissue Res 314, 119-129.-   Enkvist M. O., Hamalainen H., Jansson C. C., Kukkonen J. P., Hautala    R., Courtney M. J. and Akerman K. E. (1996) Coupling of astroglial    alpha 2-adrenoreceptors to second messenger pathways. J Neurochem    66, 2394-2401.-   Ferguson R. K. and Woodbury D. M. (1969) Penetration of 14C-inulin    and 14C-sucrose into brain, cerebrospinal fluid, and skeletal muscle    of developing rats. Exp Brain Res 7, 181-194.-   Gage F. H. (2000) Mammalian neural stem cells. Science 287,    1433-1438.-   Gallo et al. (2002) J Cell Biology 158, 731-40.-   Gerhart D. Z., Enerson B. E., Zhdankina O. Y., Leino R. L. and    Drewes L. R. (1997) Expression of monocarboxylate transporter MCT1    by brain endothelium and glia in adult and suckling rats. Am J    Physiol 273, E207-213.-   Hoffman H. J. and Olszewski J. (1961) Spread of sodium fluorescein    in normal brain tissue. A study of the mechanism of the blood-brain    barrier. Neurology 11, 1081-1085.-   Janzer R. C. and Raff M. C. (1987) Astrocytes induce blood-brain    barrier properties in endothelial cells. Nature 325, 253-257.-   Jin K., Zhu Y., Sun Y., Mao X. O., Xie L. and Greenberg D. A. (2002)    Vascular endothelial growth factor (VEGF) stimulates neurogenesis in    vitro and in vivo. Proc Natl Acad Sci USA 99, 11946-11950.-   Johanson C. E. (1989) Ontogeny and phylogeny of the blood-brain    barrier, in Implications of the blood-brain barrier and its    manipulation (Neuwelt E. A., ed.). Plenum, New York.-   Katoh (2002) Int. J. Mol. Med 10, 683-687.-   Kaufman et al., U.S. Pat. No. 7,176,023-   Kis B., Deli M. A., Kobayashi H., Abraham C. S., Yanagita T., Kaiya    H., Isse T., Nishi R., Gotoh S., Kangawa K., Wada A., Greenwood J.,    Niwa M., Yamashita H. and Ueta Y. (2001) Adrenomedullin regulates    blood-brain barrier functions in vitro. Neuroreport 12, 4139-4142.-   Kniesel U., Risau W. and Wolburg H. (1996) Development of    blood-brain barrier tight junctions in the rat cortex. Brain Res Dev    Brain Res 96, 229-240.-   Krizbai I. A. and Deli M. A. (2003) Signalling pathways regulating    the tight junction permeability in the blood-brain barrier. Cell Mol    Biol (Noisy-le-grand) 49, 23-31.-   Kubo et al. (2005) Blood, 105, 4590-4597.-   LeVine S. M. and Goldman J. E. (1988) Embryonic divergence of    oligodendrocyte and astrocyte lineages in developing rat cerebrum. J    Neurosci 8, 3992-4006.-   Louissaint A., Jr., Rao S., Leventhal C. and Goldman S. A. (2002)    Coordinated interaction of neurogenesis and angiogenesis in the    adult songbird brain. Neuron 34, 945-960.-   McCarty J. H., Monahan-Earley R. A., Brown L. F., Keller M.,    Gerhardt H., Rubin K., Shani M., Dvorak H. F., Wolburg H., Bader B.    L., Dvorak A. M. and Hynes R. O. (2002) Defective associations    between blood vessels and brain parenchyma lead to cerebral    hemorrhage in mice lacking alphav integrins. Mol Cell Biol 22,    7667-7677.-   Mi H., Haeberle H. and Barres B. A. (2001) Induction of astrocyte    differentiation by endothelial cells. J Neurosci 21, 1538-1547.-   Nico B., Quondamatteo F., Herken R., Marzullo A., Corsi P., Bertossi    M., Russo G., Ribatti D. and Roncali L. (1999) Developmental    expression of ZO-1 antigen in the mouse blood-brain barrier. Brain    Res Dev Brain Res 114, 161-169.-   Ostenfeld and Svendsen (2003) Adv Tech Stand Neurosurg, 28:3-89-   Ostenfeld T. and Svendsen C. N. (2004) Requirement for neurogenesis    to proceed through the division of neuronal progenitors following    differentiation of epidermal growth factor and fibroblast growth    factor-2-responsive human neural stem cells. Stem Cells 22, 798-811.-   Ostenfeld T., Joly E., Tai Y. T., Peters A., Caldwell M.,    Jauniaux E. and Svendsen C. N. (2002) Regional specification of    rodent and human neurospheres. Brain Res Dev Brain Res 134, 43-55.-   Palmer T. D., Willhoite A. R. and Gage F. H. (2000) Vascular niche    for adult hippocampal neurogenesis. J Comp Neurol 425, 479-494.-   Perriere N., Demeuse P., Garcia E., Regina A., Debray M., Andreux J.    P., Couvreur P., Scherrmann J. M., Temsamani J., Couraud P. O.,    Deli M. A. and Roux F. (2005) Puromycin-based purification of rat    brain capillary endothelial cell cultures. Effect on the expression    of blood-brain barrier-specific properties. J Neurochem 93, 279-289.-   Ramsauer M., Krause D. and Dermietzel R. (2002) Angiogenesis of the    blood-brain barrier in vitro and the function of cerebral pericytes.    Faseb J 16, 1274-1276.-   Risau W., Hallmann R. and Albrecht U. (1986a)    Differentiation-dependent expression of proteins in brain    endothelium during development of the blood-brain barrier. Dev Biol    117, 537-545.-   Risau W., Hallmann R., Albrecht U. and Henke-Fahle S. (1986b) Brain    induces the expression of an early cell surface marker for    blood-brain barrier-specific endothelium. Embo J 5, 3179-3183.-   Saunders N. R., Knott G. W. and Dziegielewska K. M. (2000) Barriers    in the immature brain. Cell Mol Neurobiol 20, 29-40.-   Savettieri G., Di Liegro I., Catania C., Licata L., Pitarresi G. L.,    D'Agostino S., Schiera G., De Caro V., Giandalia G., Giannola L. I.    and Cestelli A. (2000) Neurons and ECM regulate occludin    localization in brain endothelial cells. Neuroreport 11, 1081-1084.-   Schulze C. and Firth J. A. (1992) Interendothelial junctions during    blood-brain barrier development in the rat: morphological changes at    the level of individual tight junctional contacts. Brain Res Dev    Brain Res 69, 85-95.-   Segovia J., Lawless G. M., Tillakaratne N. J., Brenner M. and    Tobin A. J. (1994) Cyclic AMP decreases the expression of a neuronal    marker (GAD67) and increases the expression of an astroglial marker    (GFAP) in C6 cells. J Neurochem 63, 1218-1225.-   Shen Q., Goderie S. K., Jin L., Karanth N., Sun Y., Abramova N.,    Vincent P., Pumiglia K. and Temple S. (2004) Endothelial cells    stimulate self-renewal and expand neurogenesis of neural stem cells.    Science 304, 1338-1340.-   Sobue K., Yamamoto N., Yoneda K., Hodgson M. E., Yamashiro K.,    Tsuruoka N., Tsuda T., Katsuya H., Miura Y., Asai K. and    Kato T. (1999) Induction of blood-brain barrier properties in    immortalized bovine brain endothelial cells by astrocytic factors.    Neurosci Res 35, 155-164.-   Stewart P. A. and Wiley M. J. (1981) Developing nervous tissue    induces formation of blood-brain barrier characteristics in invading    endothelial cells: a study using quail-chick transplantation    chimeras. Dev Biol 84, 183-192.-   Stewart P. A. and Hayakawa K. (1994) Early ultrastructural changes    in blood-brain barrier vessels of the rat embryo. Brain Res Dev    Brain Res 78, 25-34.-   Tontsch U. and Bauer H. C. (1991) Glial cells and neurons induce    blood-brain barrier related enzymes in cultured cerebral endothelial    cells. Brain Res 539, 247-253.-   Weidenfeller C., Schrot S., Zozulya A. and Galla H. J. (2005) Murine    brain capillary endothelial cells exhibit improved barrier    properties under the influence of hydrocortisone. Brain Res 1053,    162-174.-   Weidenfeller, C., Svendsen, C., and Shusta, E. (2007)    Differentiating embryonic neural progenitor cells induce blood-brain    barrier properties Journal of Neurochemistry 101 (2), 555-565.    (Incorporated by reference)-   Wurmser A. E., Nakashima K., Summers R. G., Toni N., D'Amour K. A.,    Lie D. C. and Gage F. H. (2004) Cell fusion-independent    differentiation of neural stem cells to the endothelial lineage.    Nature 430, 350-356.-   Zerlin M. and Goldman J. E. (1997) Interactions between glial    progenitors and blood vessels during early postnatal corticogenesis:    blood vessel contact represents an early stage of astrocyte    differentiation. J Comp Neurol 387, 537-546.

1. A method of creating a multicellular blood-brain barrier model,comprising the step of: (a) culturing brain microvascular endothelialcells upon a permeable support in the presence of neural progenitorcells, wherein the cultured neural progenitor cells differentiate intomixtures of astrocytes, neurons, and oligodendrocytes such that amulticellular blood-brain barrier model is created.
 2. The method ofclaim 1 wherein the endothelial cells are isolated from mammalian braincapillaries.
 3. The method of claim 1 wherein the endothelial cells arederived from isolated embryonic stem cells.
 4. The method of claim 1wherein the endothelial cells form a monolayer wherein the cells areconfluent and express an initial TEER of 20-50 Ohm×cm² before exposureto the neural cells.
 5. The method of claim 4 wherein the TEER greaterthan 100 Ohm×cm² after exposure to the neural cells.
 6. The method ofclaim 5 wherein the TEER is greater than 200 Ohm×cm² after exposure tothe neural cells.
 7. The method of claim 1 wherein the neural progenitorcells are isolated from mammalian cortices.
 8. The method of claim 7wherein the neural cells are digested with at least one enzyme todissociate the cells.
 9. The method of claim 1 wherein the neuralprogenitor cells are grown as free-floating neurospheres beforedifferentiation.
 10. The method of claim 1 wherein the neural progenitorcells are pre-differentiated before exposure to endothelial cells. 11.The method of claim 1 wherein the neural cells are removed after theendothelial cells are confluent and express a TEER of at least 100Ohm×cm².
 12. A method of creating a multicellular blood-brain barriermodel, comprising the step of: (a) culturing brain microvascularendothelial cells upon a permeable support in the presence ofmultipotent neural progenitor cells, wherein the endothelial cells forma monolayer wherein the cells are confluent and express an initialtransendothelial electrical resistance (TEER) of 20-50 Ohm×cm² beforeexposure to the neural cells, wherein the multipotent neural progenitorcells further differentiate into mixtures of astrocytes, neurons, andoligodendrocytes, wherein the TEER is greater than 100 Ohm×cm² afterexposure of the endothelial cells to the differentiated neural cells andwherein the model is then capable of a TEER of greater than 100 Ohm×cm²for a period of at least 72 hours.
 13. The method of claim 12 whereinthe neural progenitor cells are pre-differentiated before exposure toendothelial cells.
 14. A blood-brain barrier model created by the methodof claim
 1. 15. A blood-brain barrier model created by the method ofclaim
 10. 16. A blood-brain barrier model created by the method of claim12.
 17. A blood-brain barrier model created by the method of claim 13.18. A blood-brain barrier model comprising three components within aliquid-containing vessel, wherein the first component comprises aconfluent layer of brain microvascular endothelial cells or embryonicstem cell-derived endothelial cells, the second component comprises apermeable membrane support, wherein the first component forms a layer onthe second component, and the third component comprises either (a)undifferentiated neural progenitor cells that are differentiated aftercontact with the first component to be a mixture of astrocytes, neuronsand oligodendrocytes or (b) neural progenitor cells that have beenpre-differentiated before contact with the first component to be amixture of astrocytes, neurons and oligodendrocytes, wherein the firstand second components form a barrier between a top and a bottom chamberof the vessel and the third component is placed in the bottom chamber ofthe vessel.
 19. The model of claim 15 wherein the endothelial cells areisolated from mammalian brain capillaries.
 20. The model of claim 15wherein the endothelial cells form a monolayer wherein the cells areconfluent and express an initial TEER of 20-50 Ohm×cm² before exposureto the neural cells.
 21. The model of claim 15 wherein the TEER isgreater than 100 Ohm×cm² after exposure to the neural cells.
 22. Themodel of claim 15 wherein the TEER is greater than 200 Ohm×cm² afterexposure to the neural cells.
 23. The model of claim 15 wherein theneural progenitor cells are isolated from mammalian cortices.
 24. Themodel of claim 20 wherein the cells are digested with at least oneenzyme to dissociate the cells.
 25. The model of claim 15 wherein theneural progenitor cells are grown as free-floating neurospheres beforedifferentiation.
 26. The model of claim 15 wherein the neural progenitorcells are pre-differentiated before exposure to the endothelial cells.27. The model of claim 15 wherein the third component has been removed.28. A blood-brain barrier model including three components within aliquid-containing vessel, comprising i) a first component comprising aconfluent layer of brain microvascular endothelial cells or embryonicstem cell-derived endothelial cells, wherein the endothelial cells forma monolayer and wherein the cells are confluent and express an initialTEER of 20-50 Ohm×cm² before exposure to the neural cells; ii) a secondcomponent comprising a permeable membrane support, wherein the firstcomponent forms a layer on the second component, and iii) a thirdcomponent comprising either (a) undifferentiated neural progenitor cellsthat are differentiated after contact with the first component to be amixture of astrocytes, neurons and oligodendrocytes or (b) neuralprogenitor cells that have been pre-differentiated before contact withthe first component to be a mixture of astrocytes, neurons andoligodendrocytes, wherein the first and second components form a barrierbetween a top and a bottom chamber of the vessel and the third componentis placed in the bottom chamber of the vessel, and wherein the TEER isgreater than 100 Ohm×cm² after exposure of the endothelial cells to thedifferentiated neural cells and wherein the model is then capable of aTEER of greater than 100 Ohm×cm² for a period of at least 72 hours.